Stress promotes RNA G-quadruplex folding in human cells

Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. In this report, we showed that the folding of putative rG4s in human cells into rG4 structures is dynamically regulated under stress. By using high-throughput dimethylsulfate (DMS) probing, we identified hundreds of endogenous stress-induced rG4s, and validated them by using an rG4 pull-down approach. Our results demonstrate that stress-induced rG4s are enriched in mRNA 3′-untranslated regions and enhance mRNA stability. Furthermore, stress-induced rG4 folding is readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in human cells and suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.


Increased rG4 formation under different stresses in U2OS and COS7 cells
RNA G-quadruplex formation was probed using starvation, cold shock, and heat shock stresses in U2OS cells and COS7 cells. Cold shock (10 °C overnight) and starvation stress affect rG4 formation similarly in both cell types. Both cell types showed slightly enhanced BG4 signals with heat shock (42 °C for 30 min).

Supplementary Figure 1. RNA G-quadruplex folding is enhanced in U2OS cells under different stresses.
From the top, no stress, starvation, cold shock, and heat shock, respectively.

Supplementary Figure 2. RNA G-quadruplex folding is enhanced in COS7 cells under different stresses.
From the top, no stress, starvation, cold shock, and heat shock, respectively.

Peroxidase-like activity of rG4-hemin complexes
Supplementary Figure 3. rG4 biotinylation scheme. When rG4s are incubated with hemin. and sensitized with H2O2, rG4s can be biotinylated in the presence of a suitable substrate (e.g., biotintyramide). On the right, the gel shift assay showing a super-shift of biotinylated rG4s in presence of streptavidin used for the biotin detection (Lanes 1-6); super-shift of a positive control (pre-biotinylated oligo), no shift of control RNAs (Lanes 8-9), and no shift of rG4 under non-biotinylating environment.
Circular dichroism (CD) characterization of rG4 formation by DMSeq-identified mRNA-derived oligos representing putative rG4 regions JASCO J815 spectropolarimeter was used to collect CD spectra. The oligonucleotides were dissolved in 150 mM K+ in T10E0.1 buffer. Quartz cuvettes (a 1 mm path length) were used with sample volumes of 200 μL to achieve a sample concentration of 5 μM. Spectra were collected in the range between 220 and 320 nm at 20 °C from three scans, and a buffer baseline was subtracted from each spectrum. CD was expressed as the difference in the molar absorption of the right-handed and left-handed circularly polarized light. An increased peak intensity of the oligo under K + environment at 260-265 nm and a trough at 240 nm, which shows a reduced peak intensity under Li + environment suggests the formation of a G4. All the selected candidates show the G4 characters in CD while the control oligo that cannot form a G4 structure shows no change in the CD behavior.
Electromobility gel shift assay and NMM staining of the candidate oligos 40 pmoles of candidate oligos (folded as above) were run in 20% non-denaturing gel electrophoresis and stained with NMM and SYBR Gold separately.

Molar elipticity (millidegrees)
Control (K + ) selectively while NMM selectively binds to parallel G4s. The ladders represent RNA size markers with molecular weight in Dalton.

Synthesis and characterization of biotinylated NMM
Common reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise stated. Tetrahydrofuran (THF) was distilled from sodium-benzophenone under an argon atmosphere. Reaction progress was monitored using analytical thin-layer chromatography (TLC) on pre-coated silica gel GF254 plates (Macherey Nagel

Tetraethylene glycol dimethanesulfonate (2)
Methanesulfonyl chloride (6 mL, 76.82 mmol) was added dropwise to a stirred solution of PEG4-di-OH 1 (4.455g, 22.59 mmol) and TEA (15 mL, 107.08 mmol) in dichloromethane (20 mL) at 0 0 C. After the addition was complete, the resulting mixture was stirred at room temperature for one day. Water was added to quench the reaction. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with brine (3 x 60 mL), dried over Na2SO4, filtered, and the solvent removed under reduced pressure to afford the desired product. Yield 7.75 g (98%).

Protoporphyrin IX dimethyl ester (11) 4
Hemin (10) (0.5 g, 0.76 mmol) and pyridine (0.5 mL) were placed in a three-necked flask, then MeOH (30 ml), CH2Cl2 (30 ml) and Mohr's salt (1.42 g, 5.1 mmol) were added. Acetylchloride (15 ml, 21.0 mmol) was gradually added under stirring and cooling, while the temperature was kept below 35 °C. The mixture was stirred for 1 h and then diluted with H2O (50 ml). The bottom organic layer was separated, washed with aqueous ammonia (25%, 30 ml), then with H2O (20 ml) and dried over anhydrous Na2SO4. The product was purified by chromatography on silica gel 60 mesh using CH2Cl2 as the eluent. M.p.: 238 -240 °C. Yield 0.29 g (65% A 100 mL three-neck-round-bottom flask was fitted with a reflux condenser and an oil-bubbler was charged with a solution of 11 (0.591 g, 1.00 mmol, 1 equiv) in DMA (20 mL, 0.05M), RuCl3·3H2O (0.131 g, 0.50 mmol, 0.5 equiv), then to this mixture NaBH4 (0.393 g, 10.40 mmol, 10.40 equiv) was added with portions for three times in less than 30 min under N2 atmosphere and then stirred at 25 °C. After 30 min, the UV-vis spectrum of a sample aliquot showed complete conversion. The mixture was flushed with Ar for 5 min, opened to the atmosphere, and concentrated to 5 mL. The result mixture was transformed into 200 mL water and extracted with 300 mL CH2Cl2. The organic phase was collected and washed for three times with water to remove DMA and Ru salts. The crude product was purified by column chromatography (60-80 mesh silica gel

Synthesis of biotinylated NMM derivatives
Preparation of b 2 NMM and b 1 NMM HBTU (98%, 0.18 g, 9.00 mmol, 2.2 equiv) was added to a solution of NMM-IX 14 (0.123 g, 0.211 mmol, 1.0 equiv) in anhydrous DMF (5 mL), which was stirred for 2 h at room temperature. To this solution was added a solution of 9·HCl (0.294 g, 0.561 mmol, 2.66 equiv.) and DIPEA (98%, 0.07 g, 0.09 mL, 0.528 mmol, 2.5 equiv) in anhydrous DMF (4 mL) and the solution mixture was stirred for 48 h at room temperature. After filtering, the filtrate was evaporated to dryness. The residual solid was dissolved in CH2Cl2 and the solution was washed with water, dried over anhydrous Na2SO4, and evaporated under reduced pressure to obtain a purple solid.